The long-term goal of this work is to elucidate the mechanisms that control the position of the mitotic spindle during development. Spindle positioning is essential for a number of developmental processes, including asymmetric divisions in which a polarized cell divides to produce daughters with different fates. The proposed project addresses the molecular mechanisms of spindle positioning during asymmetric divisions in the Caenorhabditis elegans embryo. In the C. elegans one-cell embryo, LET-99, a DEP domain containing protein of the DEPDC1 family, is localized in an asymmetric cortical band pattern by the PAR proteins. LET-99 in turn restricts the cortical localization of the positive regulators of G protein signaling, GPR and LIN-5, to certain regions of the cell cortex. G protein signaling is required for cortical pulling forces that act on astral microtubules to position the spindle, and GPR and LIN-5 associate with regulators of the microtubule motor dynein. Homologs of the PAR proteins, GPR and LIN-5 are important for polarity and spindle positioning in several different organisms. However, how GPR and LIN-5 regulate forces that position spindles is not known for any system. Further the molecular mechanism by which asymmetries of GPR and LIN-5 are generated in C. elegans remain to be elucidated. The experiments proposed in Aim 1 will help refine models for the mechanistic basis of force generation by determining how microtubule dynamics and the localization of microtubule binding proteins and motors correlates with the cortical force domains defined by LET-99 and GPR/LIN-5 localization. Live-imaging of GFP-tagged reporters will be used to examine cortical-microtubule dynamics and the localization of dynein and its regulators both at the cortex and on microtubule plus-ends. The hypothesis that the clasp family of microtubule plus-end binding proteins regulates microtubule dynamics to facilitate spindle orientation and then to tether the spindle will also be investigated, using a combination of live-imaging and genetic analysis. The goal of Aim 2 is to determine how binding of LET-99 to G) subunits affects the G protein pathway such that GPR localization is inhibited at the cortex. Quantitative analysis of immunolocalization patterns and double mutant analysis will be used to determine which components of the pathway are regulated by LET-99. Biochemical approaches will be used to determine if LET-99 affects G) activity or its association with other pathway components. Because of the conservation of pathway components, the results of these studies will be relevant to asymmetric division in many systems.